WO2005033324A1 - Biocatalyseur pour produire un acide d-lactique - Google Patents

Biocatalyseur pour produire un acide d-lactique Download PDF

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Publication number
WO2005033324A1
WO2005033324A1 PCT/JP2004/014037 JP2004014037W WO2005033324A1 WO 2005033324 A1 WO2005033324 A1 WO 2005033324A1 JP 2004014037 W JP2004014037 W JP 2004014037W WO 2005033324 A1 WO2005033324 A1 WO 2005033324A1
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lactic acid
escherichia coli
microorganism
activity
culture
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PCT/JP2004/014037
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English (en)
Japanese (ja)
Inventor
Mitsufumi Wada
Toshihiro Oikawa
Daisuke Mochizuki
Junko Tokuda
Miyuki Kawashima
Tadashi Araki
Reiko Abe
Hitoki Miyake
Hitoshi Takahashi
Hideki Sawai
Takashi Mimizuka
Takashi Morishige
Yosuke Higashi
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Mitsui Chemicals, Inc.
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Priority to BRPI0414673A priority Critical patent/BRPI0414673B1/pt
Priority to US10/573,813 priority patent/US8669093B2/en
Priority to ES04773417.3T priority patent/ES2619176T3/es
Priority to JP2005514410A priority patent/JP4473219B2/ja
Priority to EP04773417.3A priority patent/EP1669460B1/fr
Publication of WO2005033324A1 publication Critical patent/WO2005033324A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P7/00—Preparation of oxygen-containing organic compounds
    • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
    • C12P7/56—Lactic acid

Definitions

  • the present invention relates to a microorganism capable of selectively and highly producing D-lactic acid, and a method for producing D-lactic acid using the microorganism. More specifically, the present invention relates to a method for efficiently producing high-purity lactic acid, and more particularly to an efficient method for producing D-lactic acid, which produces a small amount of pyruvic acid. Further, the present invention relates to a method for producing D-lactic acid, which comprises using a microorganism in which FAD-dependent D-lactic acid dehydrogenase is inactivated or reduced.
  • the present invention also relates to a microorganism that produces D-lactic acid without producing succinic acid / fumaric acid as an impurity, and a method for producing D-lactic acid using the same.
  • Polylactic acid is the background technology biodegradable polymer, C_ ⁇ 2 problem and energy manifested as both Sustainability pyridinium tea (sustainability) issues, LCA (life cycle Asesume down Bok) garnered strong attention as the corresponding products Therefore, an efficient and inexpensive production method is required for lactic acid as a raw material.
  • polylactic acid currently industrially produced is an L-lactic acid polymer.
  • Lactic acid includes L-lactic acid and D-lactic acid, and D-lactic acid has recently attracted attention as an intermediate for polymer raw materials, agricultural chemicals, and pharmaceuticals. It is getting.
  • lactic acid as a raw material requires high optical purity.
  • microorganisms that efficiently produce lactic acid such as lactic acid bacteria and filamentous fungi, in nature, and some of the lactic acid production methods using these have already been put to practical use.
  • L actbac illus de lb rue c As microorganisms capable of efficiently producing D-lactic acid, microorganisms belonging to the genus Sporo 1 actobaci 11 us are known.
  • the accumulated amount of lactic acid reached a high level, but by-products other than lactic acid contained in the culture medium, such as acetic acid, ethanol, acetate, and pyruvic acid, could not be removed during the purification process.
  • the quality of the final product lactic acid may be reduced. It is also a serious problem that the optical purity is reduced due to the contamination of optical isomers.
  • Escherichia coli in Escherichia coli, yeast, human cultured cells, etc., which have abundant genomic information and have a good track record as genetically modified hosts, gene disruption can be performed relatively easily. Particularly, Escherichia coli is most preferable in terms of growth speed and easiness of culture. Furthermore, since the lactic acid produced by Escherichia coli is only the D-form, it is a convenient host for the purpose of obtaining D-lactic acid with high optical purity. However, wild-type Escherichia coli has low D-lactic acid productivity and produces various by-product organic acids in addition to D-lactic acid. In order to solve this problem, attempts have been made in the past to modify the metabolic pathway of Escherichia coli by genetic recombination to selectively produce high levels of D-lactic acid.
  • Chang et al. (Chang, D.—E., et. Al., Appl. Environ. Microbiol., Vol. 65 (4), PP1384-1389 (1999)) describe a phosphotransacetylase of Escherichia coli. Pta) and phosphoenol pyruvate carboxylase (hereinafter sometimes abbreviated as PPc) heavy mutants with 5% glucose and amino acids.
  • PPc phosphoenol pyruvate carboxylase
  • L-lactate dehydrogenase (hereinafter sometimes abbreviated as 11d), which is a catalytic enzyme for the reaction to generate pyruvate from the L-form, is expressed under aeration conditions. If there is a method for efficiently performing lactic acid fermentation, it is possible to produce L-forms in the culture medium by utilizing the L-forms contained in the culture medium and to produce D-forms with high optical purity. Although it is expected, there has been no technology to achieve this.
  • D-lactate dehydrogenase is classified into NADH-dependent and FAD-dependent by its difference in coenzyme dependence.
  • NADH-dependent D-lactate dehydrogenase catalyzes the reaction of pyruvate to D-lactate in vivo.
  • NADH-dependent D-lactate dehydrogenase derived from Escherichia coli is called 1 dhA.
  • Yang et al. (Yang, ⁇ . ⁇ ⁇ , et. Al., Metab. Eng., Vol. 1 (2), ppl41-152 (1999)) introduced an expression vector incorporating the 1 dh A gene into Escherichia coli. It has been reported that D-lactic acid accumulation can be improved as low as about 8 g / L. That is, it is known from the data disclosure of Yang et al. That the enhancement of 1-dhA activity in Escherichia coli improves the productivity of D-lactic acid. On the other hand, according to the report of Punch et al. [: Bunch, PK, Microbiology, Vol.
  • Examples of overexpression of D-lactate dehydrogenase (hereinafter sometimes referred to as ldh) derived from bacteria other than Escherichia coli include Cocker et al.
  • 1 dh derived from Lacto obacillus helveticus. [Kochhar, S., Eur. J. Biochem., (1992) 208, 799-805) and Lacto ob acillus bul garicus 1 dh expression examples include Cocker et al. [Kochhar, S., Biochem.
  • the D-lactic acid productivity of microorganisms having inactivated or reduced pf 1 activity and enhanced 1 dh A activity has not been well known yet.
  • the gene enrichment method using an expression vector may cause a problem that the vector is dropped, the expression level of the target gene is reduced, and the productivity of the target substance is reduced.
  • there are several problems to be solved in the method of enhancing the 1 dh gene using an expression vector when applied to industrial production of D-lactic acid and there is a need for an alternative method of enhancing the gene.
  • no report has been made on such efforts.
  • d 1 d is stored in the database of E.co 1 i Genetic Stock Center (CGSC) attached to Y a 1 e University, which is one of the institutes for substituting E. coli strains.
  • CGSC E.co 1 i Genetic Stock Center
  • pf 1 double mutants of pf 1 were searched, the results of Mat-Jan et al. (Mat-Jan, F., et. Al., J. Bacteriol., Vol. 171 (1), pp342-348 (1989)) is output as a relevant case, but as a result of actual scrutiny, this paper did not find any description of double-disrupted strains d1d and pf1.
  • D-lactic acid has achieved both productivity and selectivity at the same level as the industrial level by fermentation production using microorganisms.
  • succinic acid-fumaric acid a major by-product organic acid, was not used.
  • Patent Document 1 JP-A-11-056361
  • Non-Patent Document 1 Chang, D.-E., et. Al., Appl. Environ. Microbiol., Vol. 65 (4), ppl 384-1389 (1999)
  • Non-Patent Document 2 Zhou, S., et. Al., Appl. Environ. Microbiol., Vol. 69 (1), PP399-407 (2003)
  • Non-patent document 3 Con tag, P.R., et.al., Appl. Environ. Microbiol., Vol. 56 (12), PP3760-3765 (1990)
  • Non-patent document 4 Yang, YT "et. Al., Metab. Eng., Vol. 1 (2), ppHl-152 (1999)
  • Non-patent document 5 Bunch, PK, et. Al., Microbiology, Vol. 143 (Pt 1), pl87-195 (1997)
  • Non-Patent Document 6 ochhar, S., et. Al., Eur. J. Biochem., Vol. 208 (3), PP799-805 (1992)
  • Non-Patent Document 7 Kochhar, S., et. Al., Biochem. Biophys. Res. Vol.185 (2), pp705-712 (1992)
  • Non-Patent Document 8 Sol em, C., et. Al., Appl. Environ. Microbiol., Vol. 68 (5), PP2397-2403 (2002)
  • Non-Patent Document 9 Shaw, L., et. Al., J. Bacteriol., Vol. 121 (3), ppl047-1055 (1975)
  • Non-Patent Document 10 Barnes, E.M., et.al., J. Biol. Chem., Vol.246 (17), PP5518-5522 (1971)
  • Non-Patent Document 11 Mat-Jan, F., et. Al., J. Bacteriol., Vol. 171 (1), pp342-348 (1989)
  • Non-Patent Document 12 Courtright, J.B. et.al., J. Bacteriol., Vol. 102 (3), PP722-728 (1970)
  • Non-Patent Document 13 Dreyfus, LA, et. Al., J. Bacteriol., Vol. 136 (2), pp757-764 (1978) DISCLOSURE OF THE INVENTION
  • One of the objects of the present invention is high production of D-lactic acid.
  • Another object of the present invention is to provide a method for producing D-lactic acid, which has high optical purity and low amount of by-product organic acids, and is highly selective.
  • Another object of the present invention is to provide a method for producing D-lactic acid in which the amount of pyruvic acid that has been conventionally difficult to remove from a medium in which lactic acid is produced and accumulated by a microorganism as an impurity organic acid has been reduced, has been reduced. Is to do.
  • Another object of the present invention is to provide a method for enhancing a stable D-lactic acid dehydrogenase gene, which is an alternative to the method using an expression vector, and to provide a method for producing D-lactic acid at a higher level. .
  • the present inventors have conducted intensive studies to solve these problems, and as a result, the i-pyruvate formate lyase (Pf 1) activity was inactivated or reduced, and the NAD derived from Escherichia coli We found that bacteria with enhanced H-dependent D-lactate dehydrogenase (1 dh A) activity produced D-lactate in a shorter time than ever before, and achieved an unprecedentedly high accumulation.
  • the gene encoding 1 dh A is linked on the genome to the promoter of a gene that controls the expression of proteins involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis.
  • the microorganisms expressed in this way it is possible to produce a significant amount of D-lactic acid in a shorter time than the method of enhancing gene expression using an expression vector.
  • the amount of D-lactate dehydrogenase expressed in the cell is larger than that of the method of the present invention, but for some reason, this enzyme is not directly linked to the high production of lactic acid. It is extremely surprising that the productivity of D-lactic acid is dramatically improved even if the expression level of the enzyme in the cell is not so high as described above.
  • the inventors found that a part of pyruvic acid present in the microorganism culture solution was actually produced from d-lactic acid by d1d, and furthermore, the d1d gene was substantially impaired.
  • the microorganisms that have been activated or reduced, the growth of the microorganisms is not suppressed as compared to the host, and it contains high-quality D-lactic acid with a reduced pyruvate content in the medium. It has been found that a culture solution can be obtained.
  • the present inventors have a TCA cycle, inactivate or reduce malate dehydrogenase (mdh) activity, and inactivate or reduce aspartate ammonium lyase (asp A) activity.
  • the present invention has been accomplished by finding that by using the above microorganisms, it is possible to suppress the by-products of succinic acid and fumaric acid while maintaining high productivity of D-lactic acid.
  • the present invention is as follows.
  • a method for producing lactic acid comprising recovering lactic acid from lactic acid.
  • a bacterium with enhanced NADH-dependent ffiD-lactate dehydrogenase (I dh A) activity derived from Escherichia coli and inactivated or reduced pyruvate formate lyase (pf 1) activity is cultured and obtained.
  • a method for producing D-lactic acid comprising recovering D-lactic acid from the obtained culture.
  • pf 1 A microorganism characterized in that the activity is inactivated or reduced, and / or the activity of NADH-dependent D-lactate dehydrogenase (ldhA) derived from Escherichia coli is enhanced.
  • microorganism according to any one of [7] to [9] is cultured in a liquid medium, D_lactic acid is generated and accumulated in the culture solution, and D-lactic acid is separated from the culture solution.
  • Microorganisms whose FAD-dependent D-lactate dehydrogenase (d1d) activity has been inactivated or reduced are cultured in a liquid medium, and D-lactic acid is produced and accumulated in the culture medium.
  • a method for producing D-lactic acid comprising separating D-lactic acid.
  • NADH-dependent D-lactate dehydrogenase derived from Escherichia coli is involved in the production of proteins involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis.
  • the promoter of a gene that controls the expression of proteins involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis in Escherichia coli is a promoter of the glyceraldehyde 3-phosphate dehydrogenase gene derived from Escherichia coli. Escherichia coli described in [18].
  • [21] A method for producing D-lactic acid by culturing the microorganism according to any one of [15] to [20] using a medium.
  • mdh malate dehydrogenase
  • pf1 pyruvate formate lyase
  • [29] The method for producing lactic acid according to any one of [1] to [6], [10] to [14], [21], and [28], which is characterized by culturing under aeration conditions. .
  • the aeration conditions are for water at a temperature of 30 ° C, under conditions that allow oxygen supply that can be achieved under conditions where the oxygen transfer capacity coefficient KL & at normal pressure is 1 hi or more and 400 hi or less.
  • (31) the method according to any one of (1) to (6), (10) to (14), (21), (28) to (30), wherein the culture pH is 6 to 8.
  • a microorganism having high D-lactic acid productivity and D-lactic acid selectivity is provided. Then, by culturing the microorganism produced according to the present invention to produce D-lactic acid, it becomes possible to produce high-purity D-lactic acid more economically as compared with the existing method.
  • a bacterium which produces D-lactic acid with a small amount of pyruvic acid produced.
  • FIG. 1 is a graph showing the time-dependent change in the amount of D-lactic acid accumulated in a culture solution in Example 20.
  • the triangles indicate the results of the MG1655Apfl Ad ld strain (Example 15), and the squares indicate the results of the MG1655Apfl Ad ld / pGAP1 dhA strain (Example 18).
  • the circle shows the result of the MG1655ApflAdld / GAPp1dh genome-introduced strain (Example 19).
  • FIG. 2 is a graph showing the time-dependent change in the lactic acid accumulation concentration in the culture solution in Example 24.
  • the cross shows the results for the ⁇ f 1 ⁇ 1 d strain
  • the circles show the results for the ⁇ f 1 ⁇ 1 dAm dh strain
  • the triangles show the results for the ⁇ f 1 ⁇ 1 dApp c strain
  • the squares show the results for the ⁇ f 1 ⁇ strain.
  • the results for the d1d ⁇ frd strain are shown.
  • FIG. 3 is a graph showing the change over time of the succinic acid accumulation concentration in the culture solution in Example 24.
  • the cross indicates the results for the ⁇ pf1 ⁇ d1d strain
  • the circle indicates the results for the ⁇ pf1 ⁇ d1d ⁇ mdh strain
  • the triangle indicates the results for the ⁇ f1 ⁇ 1 ⁇ pc strain
  • the square indicates the results for the ⁇
  • the results of the f1 ⁇ d1d ⁇ frd strain are shown.
  • FIG. 4 is a graph showing the change over time in the lactic acid accumulation concentration in the culture solution in Example 25.
  • the circles show the results of the ⁇ p ⁇ 1 ⁇ d 1 d Amdh ⁇ asp strain
  • the triangles show the results of the ⁇ ⁇ f 1 ⁇ d 1 d Amdh ⁇ asp / GAP 1 dh A genome-introduced strain
  • the squares show the results.
  • the results of the ⁇ pf 1 ⁇ d 1 d Amdh strain are shown.
  • FIG. 5 is a graph showing the change over time in the concentration of fumaric acid accumulated in the culture solution in Example 25.
  • the circles show the results of the ⁇ pf 1 ⁇ d 1 d Amd h ⁇ asp strain
  • the triangles show the results of the ⁇ f 1 ⁇ 1 dAmdhA s pZGAP 1 dh A genome-introduced strain
  • the squares show the results of the ⁇ pf 1 ⁇ d
  • the results for the 1 d Amdh strain are shown.
  • the pyruvate formate lyase (pf1) in the present invention is classified into the enzyme number 2.3.1.54 based on the report of the International Union of Biochemistry (IUB) Enzyme Committee, and formate acetyl It is an enzyme also called transferase.
  • This enzyme is a generic term for enzymes that reversibly catalyze the reaction that produces formic acid from pyruvate. Inactivation in the present invention refers to a state in which the activity of the enzyme measured by an existing measurement system is below the detection limit.
  • reduction in the present invention refers to a state in which the activity of the enzyme is significantly reduced due to mutation and / or genetic recombination of the gene encoding the enzyme, compared to the state before the treatment.
  • the heterofermentative bacterium in the present invention means a bacterium capable of fermentatively decomposing sugar and producing at least one or more substances selected from formic acid, acetic acid, succinic acid, and ethanol in addition to lactic acid.
  • Escherichia coli is preferred as the heterozygous bacterium of the present invention, and as the heterofermentative bacterium in which the activity of pyrpetoformate lyase (pf1) is inactivated or reduced
  • Examples include any Escherichia coli wild-type pf1 gene-disrupted strain and Escherichia coli E. coli MT-10934 that can be prepared by the methods described in the Examples and the like.
  • the above MT—10934 is a strain in which the activity of pf1 has already been confirmed to be reduced, and the present invention can be easily carried out.
  • This strain is deposited under the deposit number FE RM BP-107, at the Patent Organism Depositary, the National Institute of Advanced Industrial Science and Technology, National Institute of Advanced Industrial Science and Technology, 1-1-1, Tsukuba, Higashi, 1-chome, Ibaraki Prefecture. It has been deposited on January 8, 2002 based on the Budapest Treaty on International Recognition of Deposits of Microorganisms. :
  • a single mutant of pf1 is a wild-type strain having an arbitrary F— property, such as MG1655, W3110, etc. because MT—10934 has the property of H fr C. After mixing for 2 hours in LB medium and LB medium, dilute to obtain a single colony and select the desired mutant.
  • the pf1 mutant has a lower amount of formic acid in anaerobic culture than the wild-type strain, and can be obtained by selecting them as an index.
  • the culture according to the present invention means culturing the microorganism according to the present invention using a medium.
  • the medium to be used should be a medium containing organic trace elements, nucleic acids, vitamins, etc. required by microorganisms to produce a carbon source, a nitrogen source, inorganic ions, and lactic acid. No restrictions.
  • the medium to which two or more amino acids are added in the present invention means a medium containing at least two or more of naturally occurring amino acids, and includes yeast extract, casamino acid, peptone, whey, molasses Also, a medium containing a hydrolyzate of a natural product such as corn steep liquor or a natural product extract is included.
  • a medium containing 0.5% to 20% of at least one selected from yeast extract, peptone, whey, molasses, corn steep liquor, or a mixture thereof is preferable. % To 15% is more preferable. Particularly, addition of corn steep liquor has a great effect. At this time, it is better not to add a salt such as ammonium sulfate.
  • the medium is usually a liquid medium.
  • the cultivation conditions vary depending on the cells and cultivation apparatus prepared.
  • the culture temperature should be 20 to 40 ° C, more preferably 25 ° C.
  • the culture is preferably performed at a pH of from 6.0 to 7.2, and the pH is preferably adjusted from 6.0 to 7.2, more preferably from 6.5 to 6.9, with NaOH, NH 3 or the like.
  • the cultivation time is not particularly limited, but is the time required for the cells to grow sufficiently and to produce lactic acid.
  • MT-10934 may produce formic acid in the pH range of pH 7 to 7.5, whereas in the pf1 gene-disrupted strain of MG1655, no production of formic acid is confirmed by the culture method of the present invention. Therefore, Escherichia coli as a heterozygous bacterium When formic acid is observed as in the case of MT-10934 when lactic acid is produced using a medium with a pH near neutral with the cells used, During production, the pH of the medium is controlled to be slightly more acidic than neutral. If no formic acid is observed as in the case of the MG1655 pf1 gene-disrupted strain, the medium must be used for the actual production of lactic acid. The maximum productivity can be obtained by controlling the pH to neutral or slightly neutral.
  • the culture in the present invention refers to bacterial cells, culture solutions, and processed products thereof produced by the above-described method.
  • a generally known method can be used from a culture solution, for example, a method of directly distilling after acidification, or a method of lactide.
  • a method of forming and distilling, a method of adding alcohol and a catalyst, esterifying and then distilling, a method of extracting in an organic solvent, a method of separating with an ion exchange column, a method of concentrating and separating by electrodialysis, and a combination thereof Method can be adopted.
  • the cells produced by the method of the present invention produce a group of enzymes suitable for producing lactic acid, it is possible to further produce and recover lactic acid by using this enzyme, and it is also necessary to produce lactic acid from the culture. Considered as part of the recovery method.
  • the NADH-dependent D-lactate dehydrogenase (ldh A) derived from Escherichia coli in the present invention is an enzyme derived from Escherichia coli that produces D-lactic acid and NAD from pyruvate and NADH.
  • An enzyme produced from a gene having a sequence contained in the above can be exemplified.
  • “enhanced 1dh A activity” means that a gene encoding 1 dh A is significantly more mutated and / or recombined than a state before the treatment thereof due to mutation and / or genetic recombination of the gene encoding 1 dh A. It refers to a state where the activity of the enzyme produced is increased.
  • the bacterium in the present invention is a general prokaryotic microorganism.
  • MT-10934 / pG1y1 described in Examples of the present invention is exemplified.
  • dh A can be exemplified. This strain is obtained by culturing a heterolactic acid-fermenting bacterium with inactivated or reduced pyruvate formate lyase (pf1) activity described in [1] above in a medium supplemented with two or more amino acids. Further, it can be suitably used for a lactic acid production method characterized by recovering lactic acid from nutrients.
  • a gene encoding 1 dh A is replaced with a promoter of a gene that controls the expression of a protein involved in glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis. It is effective to integrate the ligated state into an expression plasmid and introduce it into a desired bacterium.
  • the promoter of a gene that controls the expression of a protein involved in the glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis is a strong promoter that functions constantly in bacteria, preferably in Escherichia coli.
  • a promoter that is not easily suppressed in expression even in the presence of glucose and specifically, a dalyseraldehyde triphosphate dehydrogenase promoter, a serine hydroxymethyltransferase (g1yA) promoter.
  • the bacterium thus obtained has an increased amount of accumulated D-lactic acid as compared with the case in which the expression of 1dhA is not enhanced when producing D-lactic acid under aeration conditions, and pyruvine, an impurity, is produced. As the acid concentration decreases, the optical purity of D-lactic acid can be improved.
  • the FAD-dependent D-lactate dehydrogenase (d1d) in the present invention is an enzyme that catalyzes a reaction that produces pyruvate from D-lactic acid in the presence of oxidized flavin adenine dinucleotide as a coenzyme. Refers to a generic name.
  • the microorganism in the present invention is not particularly limited as long as it is a microorganism having a D-lactic acid-producing ability, and even a microorganism having no D-lactic acid-producing ability can be modified to have a D-lactic acid-producing ability by some modification. And microorganisms having the same.
  • the d 1 d activity in the present invention is inactivated or reduced, and or A microorganism characterized in that pf 1 activity is inactivated or reduced and / or 1 dhA activity is enhanced, such as Escherichia coli MT-109.
  • the promoter of a gene that controls the expression of a protein involved in a glycolysis system, a nucleic acid biosynthesis system, or an amino acid biosynthesis system is a strong promoter that constantly functions in a microorganism, and in the presence of glucose.
  • the promoter is less likely to be suppressed in expression, and specific examples thereof include the promoter of dariceraldehyde 3-phosphate dehydrogenase (hereinafter sometimes referred to as GAPDH) and the promoter of serine hydroxymethyltransferase.
  • the promoter according to the present invention means a site to which RNA polymerase having a sigma factor binds and initiates transcription.
  • GAP DH from U.S. origin derived from Escherichia coli is described in base number 397-440 in the base sequence information of GenBankaccessonnumberX02662.
  • the gene encoding 1 dhA is used on the genome by using the promoter of the gene that controls the expression of a protein involved in the glycolysis, nucleic acid biosynthesis, or amino acid biosynthesis.
  • a microorganism characterized by expressing pf1 activity and having inactivated or reduced pf1 activity and having inactivated or reduced dd1 or d1d activity includes Escherichia coli E. coli MT-10994. (FER M BP-10058) strain can be exemplified.
  • the Escherichia coli MT-10994 strain is expressed by operably linking the 1 dhA gene to the GAP DH promoter on the genome, and pf 1 B and d 1 d are inactivated by gene disruption. Therefore, the present invention can be easily implemented using this.
  • This strain has a deposit number of FERM BP-10058, and is located at 1-1, Higashi 1-1, Tsukuba City, Ibaraki Prefecture. Deposited on March 19, 2004 based on the Budapest Treaty on International Recognition.
  • the TCA cycle is a carbon skeleton such as sugar, fatty acid, and many amino acids. Is a metabolic pathway for the ultimate complete oxidation of, and is also known as the citrate cycle, the tricarboxylic acid cycle, and the Krebs cycle.
  • Malate dehydrogenase (mdh) in the present invention is classified into enzyme number 1.1.1.137 based on the report of the International Union of Biochemistry (I.U.B.) Enzyme Committee, and is derived from malic acid. It refers to a general term for enzymes that reversibly catalyze the reaction of producing oxa mouth acetic acid in the presence of oxidized nicotinamide adenine dinucleotide, which is a coenzyme.
  • the microorganism in which the mdh activity is inactivated or reduced, the pf1 activity is inactivated or reduced, and / or the d1d activity is inactivated or reduced, Escherichia coli MT—109 94 strains can be exemplified.
  • the strain is a heterolactic acid-fermenting bacterium in which the activity of pyrpetoformate lyase (pf1) is inactivated or reduced, as described in [1] above, in a medium supplemented with two or more amino acids.
  • the present invention can be suitably used for a method for producing lactic acid, which comprises culturing and recovering lactic acid from the obtained culture.
  • Aspartate ammonium lyase (asp A) in the present invention is classified into the enzyme number 4.3.1.1 according to the report of the International Union of Biochemistry (I.U.B.) Enzyme Committee, It is also called an enzyme.
  • This enzyme is a generic term for enzymes that reversibly catalyze the reaction to produce fumaric acid from L-aspartic acid.
  • the aeration conditions referred to here do not necessarily mean that air must pass through the culture solution.
  • the upper surface may be ventilated while the culture medium is agitated and the air layer on the culture solution is ventilated. Means that gas containing oxygen flows into the inside of the culture tank.
  • the dissolved oxygen concentration changes depending on the combination of the internal pressure, the position of the stirring blade, the shape of the stirring blade, and the stirring speed.Therefore, lactic acid productivity and the amount of organic acids other than lactic acid are used as indices as follows. Optimal conditions can be determined.
  • Another index of optimal aeration conditions is that formic acid, acetic acid, succinic acid, and ethanol produced by anaerobic cultivation of the MT-10934 strain are less than 5.OgZL, more preferably less than 1.OgZL and lactic acid. These are the aeration conditions achieved by the amount of aeration and the stirring speed as produced.
  • Another indicator of optimal aeration conditions is the concentration of L-lactic acid within 10 to 100 hours when the MT-10934 strain is cultured in a medium containing 0.3% of the optical isomer L_lactic acid. Is lower than 0.02%.
  • the aeration conditions described above do not need to be performed consistently from the beginning to the end of the culture, and favorable results can be obtained by performing them in a part of the culture process.
  • composition of the medium used for the culture is shown in Table 1 below.
  • Adekinol LG 126 0.1% This medium contains 0.34% of reduced sugars after acid hydrolysis derived from corn steep liquor, 0.31% of D-lactic acid, 0.31% of L-lactic acid, and 0 free amino acids. Contains 33% and trace amounts of various organic acids.
  • Escherichia coli MT-10934 strain was inoculated into 25 ml of LB Broth, Miller culture solution (Difco 244620) placed in an Erlenmeyer flask as a preculture, followed by stirring at 120 rpm overnight, and then 1 L The whole volume was inoculated into a culture vessel (culture apparatus BM J-01 manufactured by ABLE) containing 475 g of the medium having the above composition. The cultivation was carried out at atmospheric pressure, aeration of 0.5 V vm, stirring speed of 150 rpm, cultivation temperature of 3 It, pH 6.7 (adjusted with NaOH) until glucose was completely consumed.
  • Formic acid 1.8g / L ND ⁇ 0.lg / L
  • Acetic acid 2.g / L ND 0.lg / L
  • Ethanol 0.8g / L ND 0.lg / L
  • MG1655 was obtained as ATCC47076 from American 'Type' Culture, Collection (AT CC).
  • g 1 y A Amplified by PCR using E. coli genomic DNA as a template to obtain a promoter and SEQ ID NO: 1 and SEQ ID NO: 2 as a probe. Restriction fragment Digestion with elementary EcoRI yielded a fragment encoding the g1yA promoter of about 850 bp. Furthermore, in order to obtain a structural gene of 1 dhA, the genome DNA of Escherichia coli was used as a template, SEQ ID NO: 3 and SEQ ID NO: 4 were used as probes to amplify by PCR, and the obtained fragments were restricted.
  • Lactic acid-producing bacteria MT-10934 / pG1y1dhA strain was obtained by transforming the obtained plasmid pG1y1dhA into Escherichia coli 'coli MT-10934 strain.
  • pUC 18 can be obtained by extraction from A TCC 37253 available from American Type Culture Collection by a standard method.
  • the MT-10934 strain has been registered with the Patent Organism Depositary of the National Institute of Advanced Industrial Science and Technology (AIST) on November 8, 2002, at 1-1-1, Higashi 1-chome, Tsukuba, Ibaraki Prefecture, based on the above deposit number. Has been deposited.
  • Example 2 The lactic acid-producing bacterium MT—10934 / pG1y1dhA strain obtained in Example 2 was inoculated into 25 ml of LB Broth, Mi11er culture solution (Difco 244620) placed in an Erlenmeyer flask as a preculture. Then, the cells were cultured by the method described in Example 1. After completion of the culture, lactic acid quantification and optical purity were measured by HP LC according to a standard method. Table 3 shows the results. Table 3
  • Example 4 Cloning of the Neighboring Region of the Escherichia coli pf1 Gene
  • the entire nucleotide sequence of the genome DNA of Escherichia coli is known (GenBank accession numbe r U00096), and the pyrpeto of Escherichia coli 'coli is known.
  • the nucleotide sequence of the gene encoding formate lyase (hereinafter sometimes referred to as pf1) has also been reported (Genbank accession um ber AE 000192) 0 Gene encoding pf1 (2 , 283 bp), four oligonucleotide primers represented by SEQ ID NOS: 5, 6, 7 and 8 were synthesized.
  • the primers of SEQ ID NOs: 6 and 7 have an SphI recognition site at the 5 'end.
  • Escherichia 3UMG 1655 strain genomic DNA was prepared by the method described in Current Protocolsin Mo ecu lar Biol ogy (JonWi 1ey & Sons), and the resulting genomic DNA 1 ⁇ m was obtained.
  • Primer DN A 100 pmo 1 each Approximately 1.8 kb (hereinafter sometimes referred to as pf IB-L fragment) and about 1.3 kp (hereinafter referred to as pf 1B-R fragment) by performing PCR under normal conditions using DNA fragment was amplified.
  • This DNA fragment was separated and recovered by agarose electrophoresis, and the pf1B-L fragment was digested with HindIII and SphI, and the pf1B-R fragment was digested with SphI and PstI, respectively. Two of these digested fragments were combined with a temperature-sensitive plasmid pTH18cs1 (GenBank accession onumber AB 0 196 10) (Hashimoto-Gotoh, T., et.al., Gene, Vol. 241).
  • Example 5 Preparation of Escherichia coli MG1655 strain pf1 gene-disrupted strain
  • the plasmid ⁇ f1 obtained in Example 4 was transformed into the Escherichia coli MG1655 strain, and chloramphine was used at 30 which allows the cells to retain a temperature-sensitive plasmid.
  • Transformants were obtained by overnight culture on LB agar plates containing Enicol 1 O ⁇ gZml. The obtained transformant was cultured in LB medium at 30 for 3 hours to overnight, then appropriately diluted with LB liquid medium or physiological saline, and spread on an LB agar plate containing 10 g ml of chloramphenicol. .
  • the LB agar plate was cultured in 42 that cannot retain the temperature-sensitive plasmid, and the grown transformant was obtained as a strain in which the full-length plasmid had been integrated into the Escherichia coli genome by homologous recombination between the extragenome and the genome.
  • Genomic DNA was obtained from this strain, and PCR was performed using the DNA as a type II to confirm that the chloramphenicol resistance gene of pTHI8cs1 exists on the genome, and that the gene encoding pf1B It was confirmed that the strain had a full-length plasmid integrated into the Escherichia coli genome based on the presence of regions homologous to the 5′-side region and the 3′-side region in the genome.
  • the strain having the entire plasmid integrated into the Escherichia coli genome was inoculated into a 100-ml paffled flask containing 20 ml of LB liquid medium without clamphenicol, and cultured with shaking at 3 for 4 hours.
  • genomic DNA was obtained from the selected strain, and a ⁇ -type PCR was performed to select a strain in which the gene encoding pf1 was deleted, and this strain was named MG 1655 ⁇ pf1B strain. .
  • Example 6 Production of lactic acid by MG1655 ⁇ f1 strain using casamino acid A plurality of Erlenmeyer flasks containing 25 g of LB Brot, 1 161 ′′ culture solution (0 ⁇ fc o244620) were prepared as preculture. Lactic acid-producing bacteria MG 1655, MG 1655 ⁇ pf 1 strain, and MG 1655 ⁇ pf 1 strain, and the plasmid pG 1y 1 dhA described in Example 2 were recombined by a conventional method into MG 1655 ⁇ pf 1 / pG 1 y.
  • MG1655, MG1655 ⁇ f1, and MG1655Apfl / pGlyhdhA were separately inoculated into 25 g of a culture solution placed in an Erlenmeyer flask as a preculture, and stirred and cultured at 30 ° C and 120 rpm overnight. After that, the whole amount was separately inoculated into a 1-L culture tank (culture apparatus BMJ-01 manufactured by AB LE) containing 475 g of the medium shown in Table 6. The culture was performed at atmospheric pressure, aeration rate of 0.5 vvm, stirring speed of 300 rpm, culture temperature of 35 and pH 7.2 (adjusted with NaOH) for 24 hours. After completion of the culture, lactic acid and pyruvic acid in the obtained culture solution were measured by HP LC according to a standard method. Table 7 shows the results. Table 6 Glucose 10%
  • D-lactic acid accumulation Amount 52g / L 95g / kg medium 95g / kg medium
  • Dry cell weight 2.5g / L 2.5g / L 2.5g / L
  • Example 8 MG1655 ⁇ f1 was inoculated into 25 g of a culture solution placed in an Erlenmeyer flask as a high pre-culture of lactic acid using the MG1655 ⁇ f1 strain under a high Darcos concentration, and the mixture was inoculated at 120 r overnight. After culturing with stirring at pm, the whole amount was inoculated into cultivation tank of ABLE's culture device BM J-01 containing 475 g of the medium shown in Table 8 with the glucose concentration changed from 10% to 15%. did. Culture was performed at atmospheric pressure, aeration of 0.5 vvm, stirring speed of 300 rDm, culture temperature of 35, pH 7.2 (adjusted by NaOH). We went until the course withered. After completion of the culture, lactic acid was measured by HP LC according to a standard method. Table 9 shows the results. Table 8
  • MG 1655 ⁇ f1 was inoculated into 25 g of the culture solution in an Erlenmeyer flask as a preculture, and the mixture was stirred at 120 rpm overnight, and then placed in a culture tank of an AB LE culture device BM J-01. The whole amount was inoculated into a medium containing 475 g of a medium in which the corn steep liquor concentration shown in Table 10 was changed from 1 to 10%.
  • the culture was carried out at atmospheric pressure, aeration of 0.5 vvm, stirring speed of 300 rpm, culture temperature of 35, pH 7.2 (adjusted with NaOH). ) For 24 hours. After completion of the culture, lactic acid was measured by HPLC according to a standard method. Table 11 shows the results. Table 10
  • MG 1655 ⁇ f1B / pG1y1dhA was inoculated into 25 g of a culture solution in an Erlenmeyer flask as a preculture, followed by stirring culture at 120 rpm overnight, and then a ABLE BMJ— The total amount was inoculated into the culture tank No. 01 containing 475 g of a medium in which the concentration of corn steep liquor shown in Table 15 was changed from 1 to 10%.
  • Culture The test was carried out at atmospheric pressure, aeration rate of 0.5 vvm, stirring speed of 300 1.1, culture temperature of 35 ° C and pH 7.2 (adjusted with NaOH) for 24 hours. After completion of the culture, the measurement of D-lactic acid was performed by HPLC according to a standard method. Table 16 shows the results. Table 15
  • the 1% corn steep prima spike has the lowest productivity of all, but has an unprecedented production rate of 58 gZL in 24 hours.
  • the conversion rate of glucose used to D-lactic acid was maintained at 90% or more.
  • MG1655 ⁇ f1B / pG1y1dhA was inoculated into 25 g of a culture solution placed in an Erlenmeyer flask as a preculture, followed by stirring culture at 120 rpm overnight, and then an ABLE BMJ-01-01 culture device.
  • the total amount was inoculated into a culture tank containing 475 g of the medium shown in Table 17.
  • the culture was performed at atmospheric pressure, aeration conditions shown in Table 18, culture temperature 35, PH 7.2 (adjusted with NaOH) for 24 hours.
  • the amount of residual glucose was measured by Darcos CII-Test Co. (Wako Pure Chemical Industries). Table 17
  • Test plot 1 2 3 4 Ventilation volume (vvm) 0 0.5 0.5 0.5 0.5 0.5 0.5 0.5 Stirring speed (rpm) 200 200 400 600
  • Example 13 Preparation of Escherichia coli MG1655 Strain d1d Gene Deletion Strain CAACACCAAGCTTTCGCG (SEQ ID NO: 9) and TAC were prepared based on the genetic information of the d1d gene vicinity region of genomic DNA derived from MG1655 strain.
  • TCCACTCCTTGTGGTGGC SEQ ID NO: 10
  • AACTGCAGAA ATTACGGATGGCAGAG SEQ ID NO: 11
  • TGTTCTAGAAA PCR was performed using GTTCTTTGAC (SEQ ID NO: 12).
  • Each of the obtained fragments was digested with restriction enzymes Hindll and PstI, and PstI and Xbal to obtain fragments of about 1140 bp.
  • This fragment was transformed with the temperature-sensitive plasmid ⁇ 18cs1 (Hashimoto-Gotoh, T., et.al., Gene, Vol. 241 (1), ppl85-191 (2000)) using Hind III, XbaI.
  • DH5 ⁇ strain is transformed at 30 "C to obtain a transformant growing on an LB agar plate containing 1.0 M gZml of chloramphenicol.
  • the obtained colony was cultured overnight in an LB liquid medium containing chloramphenicol 10 / ml at 30 at 30 ° C., and the plasmid was recovered from the obtained cells.
  • Transformation was performed to obtain a transformant growing on an LB agar plate containing 10 g / ml of chloramphenicol
  • the obtained transformant was spread on an agar plate and cultured overnight at 30.
  • LB agar plate containing 1 Og / m1 of chloramphenicol was obtained these cultures.
  • the procedure of obtaining a colony growing at 42 was repeated once again, and a clone in which the entire plasmid was integrated into the chromosome by homologous recombination was selected. Has no plasmid in the cytoplasm.
  • the above clone was spread on an LB agar plate, cultured overnight at 30 ° C., inoculated into an LB liquid medium (3 ml Z test tube), and cultured with shaking at 421 for 3 to 4 hours. This was diluted as appropriate single colonies should be obtained (10 one about 2 to 10 6), plated on LB agar plates, and cultured overnight at .42, to obtain colonies. Pick up 100 colonies at random from the colonies that appeared, grow them on LB agar plates and LB agar plates containing 10 g / m1 of chloramphenicol, and grow them only on LB agar plates. A sensible crown was chosen.
  • the plasmid ⁇ f1 obtained in Example 4 was transformed into the MG 1655 Ad 1d strain to obtain a transformant growing on an LB agar plate containing chloramphenicol 10 [Ig / m1].
  • the resulting transformant was spread on an agar plate, and cultured at 30 with an agar plate. Next, these cultured cells were applied to an LB agar plate containing 10 gZml of chloramphenicol to obtain colonies growing at 42.
  • a MG1655 d1d, Pf1 gene-deleted strain was obtained from the resulting clone in the same manner as in Example 5, and named MG1655 Apfl Adld strain. [Example 16] Introduction of 1 dh A expression vector into MG 1655 Ad 1d strain and MG 1655 Apfl Ad ld strain
  • Example 2 By transforming the plasmid pG1y1dhA obtained in Example 2 into MG1655 ⁇ (11 d strain, MG1655Apfl Ad ld strain, respectively, the MG1655 ⁇ 1 d / pG1y1 dhA strain, MG1655 Ap fl Ad ld / pG lyl dhA strain was obtained.
  • Example 17 MG 1655 strain, MG 1655 Ad ld strain, MG 1655 Ap fl strain, MG 1655 um pfl Ad ld strain, MG 1655 ⁇ d 1 d / p G 1 y 1 dhA strain, MG 1655 Apfl Ad ld / MG1655, MG1655 Ad 1d, MG1655Apf1, MG1655Apfl1, MG1655Apfl Ad ld in 25 ml of LB Broth, Miller culture in a Erlenmeyer flask as a preculture.
  • MG1655AdIdZpGlyldhA strain and MG1655ApflAdIld / pGlyldhA strain were inoculated, respectively, and cultured with stirring at 120 rpm.
  • the entire amount of each preculture was transferred to a culture tank of an ABLE culture device BMJ-01 containing 475 g of a medium having the composition shown in Table 20, and cultured.
  • the culture was performed for 96 hours at atmospheric pressure, aeration rate of 0.5 vvm, stirring speed of 200 rpm, culture temperature of 31 ° C, and pH 6.7 (adjusted with NaOH).
  • the nucleotide sequence of the 1 dhA gene of Escherichia coli has already been reported (GenBank accession number U36928).
  • GPDH Chryspere aldehyde 3-phosphate dehydrogenase
  • AA and genomic DNA of Escherichia coli MG 1655 were used as templates.
  • SEQ ID NO: 14 the obtained DNA fragment was digested with a restriction enzyme EcoRI to obtain a fragment encoding a GAPDH promoter of about 100 bp.
  • a D-lactate dehydrogenase structural gene (1 dhA) the genome DNA of Escherichia coli MG1655 was prepared as a template.
  • the obtained colony was cultured overnight in an LB medium containing LB medium containing 5 OgZmL of ampicillin at 30 at LB medium, and plasmid pGAP1dhA was recovered from the obtained cells.
  • the plasmid pGAP1dhA was transformed into the MG1655 ⁇ f1; ⁇ d1d strain, and the MG1655Apfl Ad ld / pGAP1 dhA strain was obtained by overnight incubation at 37 on an LB agar plate containing ampicillin gZmL. Obtained.
  • the nucleotide sequence of the dhA gene has also been reported (GenBank cc es ssion numbe r U 36928).
  • GenBank cc es ssion numbe r U 36928 The nucleotide sequence of the dhA gene has also been reported (GenBank cc es ssion numbe r U 36928).
  • AAGGTA CCACCAGAGCGTTCTCAAGC SEQ ID NO: 17
  • GCTCTAG ATTCTCCAGTGATGTTGAATCAC SEQ ID NO: 18
  • GGT CTAGAGCAATGATTCACACGATTCG SEQ ID NO: 19
  • GGT CTAGAGCAATGATTCACACGATTCG SEQ ID NO: 19
  • GPDH Dalicellaldehyde 3-phosphate dehydrogenase
  • the fragments obtained above were digested with the restriction enzymes KpnI and XbaI, XbaI and PstI, respectively, and the fragments were digested with the temperature-sensitive plasmid pTH18cs1 with KpnI and PstI.
  • DH5 ⁇ competent cells Yukara Bio Inc.
  • LB agar plate containing 10 gXm1 of chloramphenicol. A transformant was obtained.
  • the obtained colony was cultured overnight at 30 in an LB liquid medium containing chloramphenicol 1 O / ml, and a plasmid was recovered from the obtained cells and named pTH-GAP1dhA.
  • the pTH-GAP1dhA was transformed into the MG1655ApflAdld strain at 30 and cultured overnight at 30 on an LB agar plate containing 1 Og / ml of chloramphenicol to obtain a transformant.
  • the obtained transformant was inoculated into 1 ⁇ 8 liquid medium containing 10 ⁇ 1111 of chloramphenicol, and cultured at 30 overnight.
  • these cultured cells were applied to an LB agar plate containing 10 UL g / m1 of chloramphenicil so as to obtain the cells, thereby obtaining Niguchi 21 grown at 42V.
  • the resulting colonies were cultured overnight in a drug-free LB liquid medium for 301 overnight, and further spread on drug-free LB agar plates to obtain colonies growing at 42.
  • the cultivation was performed under atmospheric pressure at an aeration rate of 0.5 V vm, a stirring speed of 200 rpm, and a culturing temperature of 35 ⁇ ⁇ 7.2 (prepared with NaOH) until glucose was depleted.
  • the amount of D-lactic acid accumulated in the obtained culture solution was measured by HPLC according to a standard method. The results are shown in Figure 1.
  • the respective D-lactic acid storage productivity was 109.0 g / L for the MG 1655 ⁇ pf 1 ⁇ d 1 d strain at 48 hours, and 115.6 g for the MG 1655Apfl Ad ld / p GAP 1 dh A strain at 48 hours.
  • One dhA genome strain was 113.5 g in 30 hours.
  • the primer having the nucleotide sequence of SEQ ID NO: 24 has a BamHI recognition site at the 5 'end, and the primer having the nucleotide sequence of SEQ ID NO: 24 has an XbaI recognition site at the 5' end.
  • the genomic DNA of Escherichia coli MG1655 strain was prepared by the method described in Cu rrent Protocol 1 sin Molecular Biol ogy (John Wi 1ey & Sons), and 1 g of the obtained genomic DNA was obtained.
  • a combination of SEQ ID NO: 21 and SEQ ID NO: 22, SEQ ID NO: 23 and SEQ ID NO: 24, and using the above primer DNAs (100 pmo1 each) under normal conditions about 800 bp (hereinafter mdh-L And a DNA fragment of about 1,000 bp (hereinafter sometimes referred to as mdh-R fragment) was amplified.
  • Plasmid pTHAmdh was transformed into Escherichia coli MG1655ApflAdId strain, and the MG1655ApflAdId strain in which the mdh gene had been broken was obtained in the same manner as in Example 5.
  • the primers of SEQ ID NOS: 26 and 27 have an XbaI recognition site at the 5 'end, and the primer of SEQ ID NO: 28 has a SacI recognition site at the 5' end.
  • DNA fragments of 450 bp (hereinafter sometimes referred to as ppc-L fragment) and about 750 bp (hereinafter sometimes referred to as ppc ⁇ R fragment) were amplified.
  • This DNA fragment was separated and recovered by agarose electrophoresis, and the ppc-L fragment was digested with HindIII and XbaI, and the ppc-R fragment was digested with XbaI and SacI, respectively.
  • the plasmid ⁇ ppc was transformed into Escherichia coli E. coli MG1655Apf1 ⁇ d1d strain, and finally the MGl655 strain ⁇ f1 ⁇ d1d strain in which the ppc gene was disrupted was obtained.
  • This strain was named MGl 655Apfl BAdldApp strain. The detailed method for obtaining this strain was in accordance with the method described in Example 5 of the present invention.
  • the entire nucleotide sequence of Escherichia coli ⁇ — mu DNA is known (GenBank accession on number U 00096), and the nucleotide sequence of the frd gene of Escherichia coli is also reported (Genb ank accession numbe). r AE 000487).
  • the frd genes to be deleted in this example are the gene encoding fr dA (1,809 bp), the gene encoding frd B (735 bp), the gene encoding fr dC (396 bp), and fr It is a gene containing four types of genes (360 bp) encoding dD.
  • oligonucleotide primers having the nucleotide sequences shown in SEQ ID NOS: 29, 30, 31 and 32 were synthesized.
  • the primer of SEQ ID NO: 29 has an EcoR I recognition site at the 5 'end
  • the primers of SEQ ID NOs: 30 and 31 have a BamHI recognition site at the 5' end
  • the primer of SEQ ID NO: 32 has an H
  • Each has an indIII recognition site.
  • frd-L fragment A DNA fragment of 00 bp (hereinafter sometimes referred to as frd-L fragment) and about 800 bp (hereinafter sometimes referred to as frd-R fragment) were amplified. This DNA fragment was separated and recovered by agarose electrophoresis, and the frd-L fragment was digested with EcoRI and BamHI, and the frd-R fragment was digested with BamHI and HindII. These two digestion fragments and the EcoRI and H of the temperature-sensitive plasmid pTHl8cs1
  • the transformant After reacting with the 1st III digest with T4 DNA ligase, the transformant was transformed into Escherichia coli DH5 ⁇ competent cells (Treasure Bio), and the 5 'upstream fragment of the frd-encoding gene and 3' A plasmid containing two fragments near the downstream was obtained, and this plasmid was named ⁇ frd.
  • Plasmid ⁇ frd was transformed into Escherichia coli E. coli MG 1655Ap fl Ad ld strain, and finally MG 1655 ⁇ pf 1 mu d 1 d strain in which the frd gene was disrupted was obtained, and MG 1655Ap f 1 ⁇ 1 ⁇ frd strain was obtained. Named. Get this stock The detailed method was in accordance with the method described in Example-5 of the present invention.
  • PCR is performed under normal conditions using 1 g of genomic DNA of Escherichia coli MG1655, SEQ ID NO: 33 and SEQ ID NO: 34, and SEQ ID NO: 35 and SEQ ID NO: 36 in combination with the above primer DNAs at 100 pmo 1 each.
  • a DNA fragment of about 910 bp hereinafter sometimes referred to as asp A-L fragment
  • pA-R fragment 1,100 bp
  • the 5 'end was phosphorylated using a T4 polynucleotide kinase by a standard method.
  • the temperature-sensitive plasmid pTHl8cs1 was digested with SmaI and then dephosphorylated with alkaline phosphatase. After reacting the two phosphorylated fragments with the dephosphorylated plasmid with T4 DNA ligase, the cells are transformed into Escherichia coli DH5 ⁇ competent cells (Takara Bio) and the gene encoding as pA A plasmid containing two fragments, a 5 'upstream fragment and a 3' downstream fragment, was obtained, and this plasmid was named pTHAasp.
  • Plasmid pTH um asp was transformed into Escherichia coli MG 1655Ap f 1 Ad 1 d m mdh strain, and finally MG 1655 ⁇ pf 1 ⁇ d 1 d Amd h strain in which the asp A gene was disrupted was obtained.
  • the plasmid pTH-GAP1dhA obtained in Example 19 was transformed into Escherichia coli K.I. MG 1655Apfl AdIdAmd ⁇ asp strain with 30, and LB agar containing kuaram fuenocol 10 [1 / ml] was used. Transformants were obtained by culturing on a plate at 3 O. The resulting transformant was inoculated into an LB liquid medium containing chloramphenicol 1 OgZm1, and cultured at 30 ° C overnight. Next, these cultured cells were applied to an LB agar plate containing 10 ag / ml of chloramphenicol to obtain colonies growing at 42. The obtained colony was cultured overnight at 30 in a LB liquid medium containing no drug, and further applied to a LB agar plate containing no drug to obtain a colony growing at 42.
  • the strain was named 1 dhA genome-introduced strain.
  • Escherichia coli M G1655 ⁇ f 1 ⁇ 1 dApp c strain, Escherichia coli MG 1655 ⁇ fl Ad l dAf rd strain obtained in Comparative Example 2 were separately inoculated, and stirred and cultured overnight at 3 O and 120 rpm. Was done. Thereafter, 475 g of the medium shown in Table 24 was placed in four 1-L culture tanks (culture apparatus BMJ-01 manufactured by ABLE), and the entire contents of the above flasks were separately inoculated. The cultivation was performed at atmospheric pressure, aeration rate of 0.5 vvm, stirring speed of 200 rpm, culture temperature of 35, and pH 7.2 (adjusted with NaOH) for 32 hours.
  • Example 25 Escherichia 'Production of D-lactic acid and fumaric acid by E. coli MG 1655Ap f 1 ⁇ d 1 dAmdhA asp strain and MG 1655 ⁇ f 1 Ad 1 dAmdhA as / GAP 1 dA genome insertion strain'
  • 25 ml of LB Broth and Mi 1 ler culture solution placed in three Erlenmeyer flasks were mixed with the Escherichia coli E. coli MGl 655Apfl Ad l dAmdhAa sp strain obtained in Example 22.
  • Example 21 coli MG1655 ⁇ f1Ad1dAmdhAasp / GAP1dhA genomic strain obtained in Example 23 and the ⁇ f1 ⁇ 1dAmdh strain obtained in Example 21 were separately inoculated. Agitation culture was performed at 120 rpm at 30 nights. Then, 475 g of the medium shown in Table 24 was placed in three 1-L culture tanks (culture apparatus BMJ-01 manufactured by ABLE), and the entire contents of the flasks were separately inoculated. The culture was carried out at atmospheric pressure, aeration rate of 0.5 vvm, stirring speed of 200 rpm, culture temperature of 35 ⁇ , pH 7.2 (adjusted with NaOH) for 48 hours. After completion of the culture, the concentrations of lactic acid and fumaric acid in the obtained culture solution were measured by HP LC according to a standard method. Figure 4 shows the results of lactate accumulation and Figure 5 shows the results of fumaric acid accumulation.
  • the Ap fl Ad l dAmdhAa sp strain accumulated at 91 gZL at 48 hours and the ⁇ f1 ⁇ 1 dAmdh strain accumulated at 48 hours at 90 gZL, whereas ⁇ f1 Ad 1 dAmdhA
  • the asp / GAP 1 dhA genome-introduced strain showed an accumulation of 98 gZL in 24 hours.
  • the ⁇ f 1 Ad 1 dAmdh strain showed accumulation of 037 g ZL at 48 hours, whereas the ⁇ f 1 Ad 1 dAmdhAa sp strain and ⁇ f 1 ⁇ 1 dAmdhAa sp / GAP 1
  • the dhA genome-introduced strain showed an accumulation of 0.01 g / L in 48 hours.

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Abstract

L'invention concerne un procédé pour produire un acide D-lactique, ainsi qu'un procédé pour produire de manière sélective un acide D-lactique présentant une grande pureté optique avec la formation de petits acides organiques utilisés en tant que sous-produits. La production de l'acide D-lactique comprend la mise en culture d'un micro-organisme présentant une activité pyruvate-formate-lyase inactivée ou diminuée et une activité élevée de l'acide D-lactique déshydrogénase (ldhA) dépendant de NADH et à origine Escherichia coli, d'un micro-organisme présentant une activité inactivée ou diminuée de l'acide D-lactique déshydrogénase (ldhA) dépendant de FAD, ou d'un micro-organisme présentant un cycle TCA, une activité inactivée ou diminuée de l'acide malique déshydrogénase et une activité inactivée ou diminuée de l'acide aspartique ammonia-lyase. L'activité de la ldhA est augmentée par liaison d'un gène codant la ldhA avec un promoteur d'un gène contrôlant l'expression d'une protéine, laquelle participe au système de glycolyse, un système de biosynthèse d'acides nucléiques ou un système de biosynthèse d'aminoacides, ou un génome.
PCT/JP2004/014037 2003-09-30 2004-09-17 Biocatalyseur pour produire un acide d-lactique WO2005033324A1 (fr)

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BRPI0414673A BRPI0414673B1 (pt) 2003-09-30 2004-09-17 escherichia coli recombinante, bem como método para a produção de ácido d-láctico
US10/573,813 US8669093B2 (en) 2003-09-30 2004-09-17 Biocatalyst for production of D-lactic acid
ES04773417.3T ES2619176T3 (es) 2003-09-30 2004-09-17 Biocatalizador para producir ácido D-láctico
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Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7326550B2 (en) 1997-09-12 2008-02-05 Tate & Lyle Ingredients Americas, Inc. Yeast strains for the production of lactic acid
US7473540B2 (en) 2005-09-22 2009-01-06 Tate & Lyle Ingredients Americas, Inc. Methods for selecting a yeast population for the production of an organic acid and producing an organic acid
WO2010032697A1 (fr) 2008-09-16 2010-03-25 三井化学株式会社 Bactérie capable de produire de l’acide lactique et méthode de production d’acide lactique
WO2010032698A1 (fr) 2008-09-16 2010-03-25 三井化学株式会社 Procédé pour la fabrication d'acide lactique à partir d'une matière première d'origine végétale et d’une bactérie produisant de l'acide lactique
WO2011013721A1 (fr) 2009-07-28 2011-02-03 三井化学株式会社 Procédé de production d'acide lactique
WO2011012693A1 (fr) 2009-07-30 2011-02-03 Metabolic Explorer Méthylglyoxal synthétase (mgs) mutante pour la production d'un agent biochimique par fermentation
JP5149619B2 (ja) * 2005-05-25 2013-02-20 サントリーホールディングス株式会社 ｐＨ制御によるコーヒー生豆の処理方法
JP2013252057A (ja) * 2012-02-24 2013-12-19 Mitsui Eng & Shipbuild Co Ltd モーレラ属細菌の遺伝子組換え法
WO2014017469A1 (fr) * 2012-07-23 2014-01-30 三井化学株式会社 Procédé de production d'acide d-lactique, procédé de production d'un polymère, et polymère
US9120891B2 (en) 2008-06-24 2015-09-01 Lg Chem, Ltd. Method for preparing polylactate and copolymer thereof using a mutant microorganism with enhanced polylactate, and the copolymer producing capability thereof

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US7326550B2 (en) 1997-09-12 2008-02-05 Tate & Lyle Ingredients Americas, Inc. Yeast strains for the production of lactic acid
JP5149619B2 (ja) * 2005-05-25 2013-02-20 サントリーホールディングス株式会社 ｐＨ制御によるコーヒー生豆の処理方法
US7473540B2 (en) 2005-09-22 2009-01-06 Tate & Lyle Ingredients Americas, Inc. Methods for selecting a yeast population for the production of an organic acid and producing an organic acid
US9120891B2 (en) 2008-06-24 2015-09-01 Lg Chem, Ltd. Method for preparing polylactate and copolymer thereof using a mutant microorganism with enhanced polylactate, and the copolymer producing capability thereof
US8614076B2 (en) 2008-09-16 2013-12-24 Mitsui Chemicals, Inc. Bacterium capable of producing lactic acid, and method for producing lactic acid
WO2010032698A1 (fr) 2008-09-16 2010-03-25 三井化学株式会社 Procédé pour la fabrication d'acide lactique à partir d'une matière première d'origine végétale et d’une bactérie produisant de l'acide lactique
JPWO2010032697A1 (ja) * 2008-09-16 2012-02-09 三井化学株式会社 乳酸生産細菌及び乳酸生産方法
WO2010032697A1 (fr) 2008-09-16 2010-03-25 三井化学株式会社 Bactérie capable de produire de l’acide lactique et méthode de production d’acide lactique
US8679800B2 (en) 2008-09-16 2014-03-25 Mitsui Chemicals, Inc. Method for producing lactic acid from plant-derived raw material, and lactic-acid-producing bacterium
JP5210391B2 (ja) * 2008-09-16 2013-06-12 三井化学株式会社 乳酸生産細菌及び乳酸生産方法
JP5243546B2 (ja) * 2008-09-16 2013-07-24 三井化学株式会社 植物由来原料から乳酸を生産する方法及び乳酸生産細菌
WO2011013721A1 (fr) 2009-07-28 2011-02-03 三井化学株式会社 Procédé de production d'acide lactique
US9096874B2 (en) 2009-07-28 2015-08-04 Mitsui Chemicals, Inc. Method for producing lactic acid under pressure that exceeds normal atmospheric pressure
JPWO2011013721A1 (ja) * 2009-07-28 2013-01-10 三井化学株式会社 乳酸製造方法
WO2011012693A1 (fr) 2009-07-30 2011-02-03 Metabolic Explorer Méthylglyoxal synthétase (mgs) mutante pour la production d'un agent biochimique par fermentation
JP2013252057A (ja) * 2012-02-24 2013-12-19 Mitsui Eng & Shipbuild Co Ltd モーレラ属細菌の遺伝子組換え法
WO2014017469A1 (fr) * 2012-07-23 2014-01-30 三井化学株式会社 Procédé de production d'acide d-lactique, procédé de production d'un polymère, et polymère
JPWO2014017469A1 (ja) * 2012-07-23 2016-07-11 三井化学株式会社 Ｄ−乳酸の生産方法およびポリマーの生産方法

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EP1669460B1 (fr) 2016-12-21
EP1669460A1 (fr) 2006-06-14
BRPI0414673B1 (pt) 2019-12-17
EP1669460A4 (fr) 2011-06-15
ES2619176T3 (es) 2017-06-23
BRPI0414673A (pt) 2006-11-28
JPWO2005033324A1 (ja) 2006-12-14
JP4473219B2 (ja) 2010-06-02
US8669093B2 (en) 2014-03-11
US20070065930A1 (en) 2007-03-22

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